Japanese encephalitis (JE) is caused by a small RNA virus belonging to the Flavivirus genus and is the most common cause of viral encephalitis worldwide. The disease principally strikes children and teen-agers and occurs in populations throughout Asia residing in rural, rice-breeding areas. Cases and deaths from JE have also occurred among travelers to Asia from the US, Europe and Australia and in military personnel serving in Asia. There is no available treatment for JE and because of the high incidence of this disease, preventable vaccination has been a high priority for many countries in Asia. Formalin-inactived vaccines produced from infected mouse brain tissue has been used in Asia and proven to be safe and efficacious. Because of the cost the vaccine is used for routing immunization in only the most affluent countries. In addition, adverse events are reported in 20% of recipients in Western countries, including severe allergic reactions in up to 1% of those vaccinated. The WHO has identified JE as a vaccine development priority. To this end, we have genetically engineered a full-length cDNA clone of Japanese encephalitis virus (JEV) from which infectious virus can be recovered (J. Virol. 66:5425,92). This clone will be used to engineer an attenuated JEV. X-ray crystallographic analysis of the tick-borne encephalitis virus envelope protein (E) has identified three major structural domains in the protein which interact to form the monomer. Computer modeling of the JEV E protein has identified potential attenuating loci in regions I and II of the E protein which are believed to function in antibody neutralization, hemagglutination and fusion and in which the attenuating mutation at E position 138 is located. Based on this model, we have identified basic amino acids in regions I and II which will be changed by mutagenesis of the cDNA clone to produce mutant virus that can be tested for virulence, immunogenicity and antigenic structure.